eclipse ti2 e confocal laser scanning microscope  (Nikon)

Journal: Nature Communications

Article Title: Vascular network-inspired fluidic system (VasFluidics) with spatially functionalizable membranous walls

doi: 10.1038/s41467-024-45781-3

Figure Lengend Snippet: a Schematic showing the printing and the self-assembly process of channels. The print nozzle is translated within anionic polyacrylamide (APAM) matrix to deposit chitosan ink against the bottom surface of a Petri dish. Driving by the electrostatic forces between chitosan, APAM, and Petri dish carrying charged groups, channel-like chambers generate with self-assembled chitosan/APAM membrane walls, which are attached to the dish surface. b (i) Confocal laser scanning microscope image showing self-assembled chambers with hollow liquid cores and thin walls. 0.02 wt% fluorescein isothiocyanate-labeled chitosan (FITC-chitosan) is mixed in the chitosan ink for visualization. (ii) Scanning electron microscope image showing the ultrathin and dense self-assembled wall. The membranous wall is freeze-dried before observation. The scale bar is 500 nm. c Soft and elastic channel walls deform under external force and resume the original shape upon removal of the force. The external force is applied by using a pipette tip. The scale bar is 1 mm. d Photographs of 3D-printed channels with arbitrary architectures, including (i) a spiral-shaped chamber, (ii) a grid-shaped chamber, and (iii) a branched network. Scale bars here are 5 mm. e Removal of matrix and infusion of liquids into a vascular network-shaped channel. The channel remains intact during the removal of matrix and ink. The channel is exposed to the air when infused with rhodamine 6 G (R6G, a red dye) aqueous solution. The scale bar is 1 cm. f Time-series optical images showing intracavitary volume of the channel can alter in response to the changed liquid volume inside. The channel with soft walls collapses as liquids inside have been partially removed via the channel outlet. The height of the channel increases from 780 to 1560 μm when the liquid perfusion rate increases from 0 to 3 mL h –1 . The height decreases to 970 μm when liquids inside partially flow out of the channel. The liquid for perfusion is an aqueous RhB solution. The scale bar is 2 mm.

Article Snippet: The cross-sectional views of polymer aggregation on the liquid interface were observed with another confocal laser scanning microscope (Eclipse Ti2-E, Nikon).

Techniques: Membrane, Laser-Scanning Microscopy, Labeling, Microscopy, Transferring

Journal: Nature Communications

Article Title: Vascular network-inspired fluidic system (VasFluidics) with spatially functionalizable membranous walls

doi: 10.1038/s41467-024-45781-3

Figure Lengend Snippet: a Fluorescence microscope images showing the membrane selectivity for different-sized molecules. Aqueous solutions of fluorescent molecules with different hydrodynamic radii ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${R}_{h}$$\end{document} R h ) are infused into channels at a flow rate of 0.5 mL h –1 . Channel walls under view are immersed under 200 μL deionized water. Fluorescein isothiocyanate-labeled dextran with a molecular weight of 4000 g mol –1 (FITC-Dex4k, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${R}_{h}$$\end{document} R h ≈ 1.4 nm) and 10,000 g mol –1 (FITC-Dex10k, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${R}_{h}$$\end{document} R h ≈ 1.9 nm) are observed passing through the channel wall within 30 min and 60 min. Trans-wall transport of fluorescein isothiocyanate-labeled dextran with a molecular weight of 40,000 g mol –1 (FITC-Dex40k, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${R}_{h}$$\end{document} R h ≈ 3.0 nm) or 70,000 g mol –1 (FITC-Dex70k, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${R}_{h}$$\end{document} R h ≈ 3.6 nm) is not observed within 60 min. Relative fluorescence intensities in images in arbitrary units (a.u.) are analyzed with MATLAB. The scale bar is 1 mm. b Localized trans-wall exchange between liquids inside and the solution deposited outside. c Spatiotemporal regulation of fluid components via the localized trans-wall introduction and extraction. (i) A Y-shaped channel is perfused with rhodamine 6 G (red dye) and T/Thioflavin T (yellow dye) aqueous solutions. (ii) Dyes at random positions and times can be extracted by depositing one deionized water droplet next to the channel. (iii) Methylene blue (MB, a blue dye) can be introduced into the channel at random positions and times by placing an MB droplet next to the channel. The scale bar is 1 cm. d Temporal regulation of MB concentration inside the channel via localized trans-wall MB transport. The channel is perfused with deionized water at a flow rate of 3 mL h –1 . After placing a 5 μL MB droplet beside the channel, liquids inside are collected 1 cm downstream from the droplet position every 2 min to analyze the MB concentration inside channel. MB concentrations with different dynamics are presented when (i) different concentrated MB droplets are placed beside the channel, or (ii) channels with different wall thicknesses are applied. Wall thickness here refers to the thickness of hydrated membrane walls. Source data are provided as a Source Data file.

Article Snippet: The cross-sectional views of polymer aggregation on the liquid interface were observed with another confocal laser scanning microscope (Eclipse Ti2-E, Nikon).

Techniques: Fluorescence, Microscopy, Membrane, Labeling, Molecular Weight, Extraction, Concentration Assay

Journal: Nature Communications

Article Title: Vascular network-inspired fluidic system (VasFluidics) with spatially functionalizable membranous walls

doi: 10.1038/s41467-024-45781-3

Figure Lengend Snippet: a (i, ii) Schematic showing the localized immobilization of functional materials on channel walls by attaching solutions to the channel. The large-sized solutes (radius or \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${R}_{h}$$\end{document} R h ≥ 3.0 nm) carrying electrostatic charges cannot penetrate membrane walls but can be electrostatically immobilized on the membranes. (iii, iv) Schematics showing the immobilization of negatively or positively charged solutes on channel walls; Confocal laser scanning microscope images showing channel walls immobilized with the positively charged FITC-labeled horseradish peroxidase (FITC-HRP, green) or negatively charged RhB-labeled glucose oxidase (RhB-GOx, red). Scale bars are 200 μm. b (i) Schematic and (ii) fluorescence microscope images showing the spatially arranged immobilization of the two enzymes on the channel walls. RhB-GOx-coated regions are visualized under RhB channel of the microscope, and FITC-HRP-coated regions are visualized under FITC channel of the microscope. Fluorescence images under RhB channel and FITC channel are collected, respectively, and merged into one. To obtain the three RhB-GOx-coated regions, region R1 is treated with 0.5 μL, 10 mg mL –1 RhB-GOx solution; region R2 is treated with 0.5 μL, 4 mg mL –1 RhB-GOx solution; region R3 is coated with 1 μL, 1 mg mL –1 RhB-GOx solution. The left HRP-coated region is treated with 0.5 μL, 1 mg mL –1 FITC-HRP aqueous solution, and the right region is coated with 1 μL, 4 mg mL –1 FITC-HRP aqueous solution. The scale bar is 2 mm. (iii) RhB-GOx-coated regions treated with different concentrations of RhB-GOx solutions have different fluorescence intensities. The relative fluorescence intensities in arbitrary units (a.u.) are obtained by analyzing fluorescence microscope images under RhB channel. Source data are provided as a Source Data file.

Article Snippet: The cross-sectional views of polymer aggregation on the liquid interface were observed with another confocal laser scanning microscope (Eclipse Ti2-E, Nikon).

Techniques: Functional Assay, Membrane, Laser-Scanning Microscopy, Labeling, Fluorescence, Microscopy

Journal: Nature Communications

Article Title: Vascular network-inspired fluidic system (VasFluidics) with spatially functionalizable membranous walls

doi: 10.1038/s41467-024-45781-3

Figure Lengend Snippet: a Schematic showing a cascade process of glucose absorption, glucose degradation, and carbon dioxide (CO 2 ) exhalation in in-vivo vascular networks. b Schematic showing a VasFluidic channel with compartmentalized functional domains, which enable glucose absorption, glucose degradation, and CO 2 exhalation, respectively. c (i) Schematic showing the upstream channel takes in glucose selectively from a solution mixture of starch and glucose. The branch channel is perfused with deionized water at a 1.5 mL h –1 flow rate. 2 μL starch-glucose aqueous solution containing 20 wt% starch and 20 wt% glucose is attached to the upstream channel. Liquid samples are then collected in the 1 cm downstream channel every 6 min. By analyzing glucose and starch concentrations in collected samples with corresponding assay kits, respectively, (ii) the trans-wall amount of glucose and starch within 30 min and (iii) the real-time glucose concentration of liquids inside the channel can be derived. d (i) Schematic showing an enzyme-mediated cascade reaction to decompose glucose, which can be visualized by a red fluorescence probe. (ii) Schematic and (iii) fluorescence microscope images showing the midstream decomposition of glucose into D-gluconolactone. The midstream channel with a GOx-coated region and an HRP-coated region converts glucose and Amplex Red into D-gluconolactone and resorufin, respectively. The scale bar is 1 mm. e (i) Schematic showing the downstream exhalation of CO 2 via trans-wall transport. The CO 2 is produced with sodium bicarbonate (NaHCO 3 ) and hydrochloric acid (HCl). (ii) Real-time CO 2 concentration outside the downstream channel. The CO 2 concentration remains unchanged when the channel is perfused with HCl aqueous solution only (0–300 s). The CO 2 concentration increases as we perfuse the channel with HCl and NaHCO 3 aqueous solutions simultaneously (300–600 s). Source data are provided as a Source Data file.

Article Snippet: The cross-sectional views of polymer aggregation on the liquid interface were observed with another confocal laser scanning microscope (Eclipse Ti2-E, Nikon).

Techniques: In Vivo, Functional Assay, Starch, Concentration Assay, Derivative Assay, Fluorescence, Microscopy, Produced

Journal: Viruses

Article Title: Neutralizing and Enhancing Epitopes of the SARS-CoV-2 Receptor-Binding Domain (RBD) Identified by Nanobodies

doi: 10.3390/v15061252

Figure Lengend Snippet: Purified 6× His-tagged nanobodies of E. coli clones 103, 105, 114, and 278 (VH103, VH105, VH114, and VH278, respectively) and their antigen binding: ( A ) Purified nanobodies from inclusion bodies of the vh -pET23b+ plasmid-transformed NiCo21 (DE3) E. coli clones 103, 105, 114, and 278 (lanes 1–4, respectively). M, protein standard marker. The numbers on the left are protein masses in kDa. ( B ) Indirect ELISA for testing the binding of 6× His-tagged VH103, VH105, VH114, and VH278 to recombinant Wuhan, Delta, and Omicron RBDs, using BSA as a control antigen. ( C – E ) The 6× His-tagged VH103, VH105, VH114, and VH278, respectively, bound to native S1 subunits of spike proteins of SARS-CoV-2 Wuhan wildtype and Delta and Omicron variants, as determined by confocal microscopy. Nanobodies stained red; native S1 subunits of the SARS-CoV-2 spike protein stained green; nuclei stained blue; co-localized VHs and S1 subunits in merged panels stained orange/yellow. ( F ) Half-maximal effective concentrations (EC50) of VH103, VH105, VH114, and VH278 against the recombinant S1 subunit.

Article Snippet: Secondary antibodies, including 1:400 dilutions of Alexa Fluor Plus 488 goat anti-rabbit IgG (Invitrogen) and Alexa Fluor Plus 555 goat anti-mouse IgG (Invitrogen), were added to the cells and kept at 4 °C for 1 h. The cells were washed with PBS, and their nuclei were stained with DAPI (Invitrogen) for 10 min. After washing, the cover slips were mounted, and the cells were examined using laser scanning confocal microscopy (Nikon C2+ Eclipse Ti2-E Laser Confocal Microscope, Nikon, Melville, NY, USA).

Techniques: Purification, Clone Assay, Binding Assay, Plasmid Preparation, Transformation Assay, Marker, Indirect ELISA, Recombinant, Control, Confocal Microscopy, Staining